Study On The Quality Standard Of Dioscorea Nipponica Makino And The Protective Mechanism Of Dioscin On Oxygen-glucose Deprivation/Reoxygenation

Research background and purpose:Dioscorea nipponica Makino is mainly produced in Liaoning,Jilin,Heilongjiang,Hebei,Shaanxi and other places.Its main active ingredient is a steroidal saponin,and it also contains polysaccharides,starch,amino acids,and flavonoids.It has the functions of regulating immunity,reducing glucose and improving cardiovascular Function,hepatoprotective,antitumor,anti-inflammatory and other pharmacological effects.It is a basic medical chemical raw material for the synthesis of steroid hormone drugs and anti-cardio-cerebral vascular disease saponin drugs.It can be used to treat rheumatoid arthritis,cardiovascular and cerebrovascular diseases.Occupying an important position in the clinical practice of TCM and the steroid drug industry,it has great application value and broad market potential.The purpose of this paper is to study the quality of medicinal herbs,to form a standard for processing methods of medicinal herbs,to improve the quality of medicinal herbs,and to construct the fingerprint of medicinal herbs.In addition,the protective mechanism of dioscin,the main component of androgenosaurus,against cerebral ischemia-reperfusion injury was explained,which provided a theoretical basis for the further development of androgenosaurus.Materials and Methods:In this paper,thin-layer identification,extractive investigation,ash investigation,moisture investigation,and dioscin content determination were conducted on the medicinal herbs of Andrographis paniculata.A method was established for the simultaneous determination of protodioscin,protogracillin,methyl protodioscin,pseudoprotodioscin and dioscin.The contents of raw protodioscin,protogracillin,methyl protodioscin,pseudoprotodioscin and dioscin in the medicinal materials of Pangosaurus from different places were compared,and the fingerprints of the medicinal materials of Pangosaurus was studied by using stoichiometry.Different processing methods were used to treat the original medicinal materials of andrographis paniculata,and the determination and comparison of protodioscin,protogracillin,methyl protodioscin,pseudoprotodioscin and dioscin were conducted.The virtual screening technology was used for reverse virtual screening of dioscin,the main substance of pangosaurus,as complement.The molecular docking was used to screen the receptor proteins,and the screening results were scored to screen the proteins associated with cerebral ischemia-reperfusion.In the study of dioscin on cerebral ischemia-reperfusion injury,in vitro studies,the chemical hypoxia inducer cobalt chloride（CoCl₂）was selected as the hypoxia inducer of PC12 cells,and the CCK-8 method was used.The optimal conditions for establishing an OGD/R model by CoCl₂ and the protective effect of dioscin on the OGD/R model of PC12 cells were determined.The autophagy-inducing activity of dioscin on ischemia-reperfusion injury in vitro and in vivo was also examined by fluorescent quantitative PCR and Western blot.Result:1.34 batches of medicinal herbs from different origins meet the pharmacopoeial extract,ash,moisture and dioscin content standards;the high-performance liquid fingerprint of the andrographis paniculata and the simultaneous determination of multi-component chromatography methods have been established,and 21 common peaks have been marked,in which There are 5 components of protodioscin,protogracillin,methyl protodioscin,pseudoprotodioscin and dioscin.The content ranges from 7.90 to33.38 mg/g,1.43 to 11.04 mg/g,2.32 to 12.05 mg/g,0.2112¹.8124 mg/g,4.815¹1.96mg/g,the median is 25.93,7.10,8.70,0.55,9.21 mg/g,and the content limits are tentatively not lower than 25.00,7.00,8.00,0.50,9.00 mg/g;similarity analysis and cluster analysis were performed,and the results of cluster analysis and similarity analysis showed that the fingerprint of P.panicosaurus was reliable;the principal component analysis used 5 main components to comprehensively evaluate the medicinal materials of P.paniculata and the results of comprehensive scores Correlation analysis,eigenvalues,and variance contribution rate further verify that protodioscin,protogracillin,methyl protodioscin,pseudoprotodioscin and dioscin can be used as new quality standard indicators.2.The sample analysis results of different processing methods show that there are differences in the content of 5 saponins of Pangosaurus.Among them,the content of protodioscin and methyl protodioscin is higher than that after steaming and freeze-drying.Steaming is higher than other processing methods.The content of pseudoprotodioscin is higher than that of dryness after steaming,freeze-drying and blast drying,while the content of dioscin is lower than other processing methods.3.Virtual screening results showed that dioscin has a high binding rate with HetR,Genome polyprotein,Beta-elicitin cinnamomin,Cold shock domain-containing protein E1,Glutaryl-CoA dehydrogenase,mitochondrial and other proteins.The top 20 protein crystals are shown in the table.3-1.The screening results are basically consistent with the reports on the pharmacological effects of dioscin.4.In the experiment of antioxidant glucose deprivation/reoxygenation injury,compared with the OGD/R model group,dioscin can significantly increase the viability of PC12 cells（p<0.01）.The mechanism may be to inhibit Cited2 and reduce LC3-Ⅱ.Protein expression level,thereby inhibiting the autophagy signaling pathway;further inhibiting Cited2,Nix and LC3 gene expression,negative feedback has to increase FoxO3 gene expression.Conclusion:1.Construction of the fingerprint of medicinal herbs and simultaneous determination of multi-component chromatography method,combined with stoichiometric analysis,the results show that the protodioscin,protogracillin,methyl protodioscin,pseudoprotodioscin and dioscin can jointly build a new quality standard.2.The traditional saponin,freeze-drying,steam-drying and drying at different temperatures and different processing methods of Pangosaurus medicinal materials were measured and compared with 5 saponins.The results showed that freeze-drying was the optimal processing method for retaining the five saponins in Dioscorea nipponica decoction pieces.3.Computer reverse virtual screening of dioscin,the results showed that Citde2could be one of the potential medicinal targets of dioscin,and related articles reported that its mechanism of action may be related to autophagy.4.Dioscin increased the expression of FoxO3 mRNA,reduced the expression of Cited2,Nix and LC3,and the protein expression of Cited2 and LC3-Ⅱ,thereby inhibiting cell autophagy caused by the OGD/R model and maintaining cell proliferation.